A l k y l a t i n g E s t e r s VII. T h e M e t a b o l i s m of I s o - P r o p y l M e t h a n e s u l p h o n a t e and I s o - P r o p y l Iodide in the Rat

200

T h e s t r u c t u r a l s i m i l a r i t y b e t w e e n d i m e t h y l m y l e r a n (I) a n d iso-propyl m e t h a n e s u l p h o n a t e ( IMS, I I , w h i c h h a s b e e n t e r m e d ~ ' H a l f - d i m e t h y l m y l e r a n ' ) , h a s led to t h e a s s u m p t i o n e t h a t t h e y s h a r e t h e s a m e i n v i v o m e c h a n i s m of ac t ion , o n e o f u n i m o l e c u l a r a k l y l a t i o n , w h i c h a c c o u n t s for t h e i r s i m i l a r b io log i ca l e f f ec t s on s p e r m a t o g e n e s i s s a n d

180

-r

g 160i

140

4 8 12 Days after treatment

120

538 Specialia EXPERIENTIA 29/5

Fig. 1. Weight responses of immature rats to iso-propyl methane- sulphonate (IMS) and dimethylmyleran. Single i.p. doses of the compounds in arachis oil were given on day 0. A--A, dimethyl- myleran 1 • mg/kg; O- - �9 IMS 1• mg/kg; X- -X , IMS 1 x 500 mg/kg; O - - 0 , controls; 1", death.

CH a C H c i t e cFI 2 CH CH 3 CH a CH CH~ / / I OSO~ CH a OSOeCH a OSO2CH a

I II

t h e h a e m o p o i e t i c s y s t e m L I n a s s e s s i n g t h e a c t i o n of a l k y l a t i n g a g e n t s o n t h e h a e m o p o i e t i c s y s t e m , E l s o n i n t r o d u c e d 4 a c l a s s i f i c a t i o n b a s e d o n t h e g r o w t h c u r v e s of r a t s t r e a t e d w i t h l e t h a l doses . C o m p o u n d s a c t i n g b y a u n i m o l e c u l a r m e c h a n i s m c a u s e r a p i d d e a t h d u r i n g t h e s i n g l e i n i t i a l w e i g h t loss p h a s e , a n d t h o s e a c t i n g b y a b i m o l e c u l a r p r o c e s s r e s u l t in d e l a y e d d e a t h d u r i n g t h e s e c o n d p e r i o d of w e i g h t loss. I n o u r s t u d i e s w i t h m e t h a n e - s u l p h o n a t e e s t e r s , we e x a m i n e d t h e w e i g h t r e s p o n s e s of r a t s to b o t h d i m e t h y l m y l e r a n a n d I M S , a n d t h e r e s u l t s ( F i g u r e 1) s u g g e s t t h a t , b y t h i s c r i t e r i on , t h e m e c h a n i s m s of a c t i o n of t h e s e 2 c o m p o u n d s m i g h t be d i f f e r e n t . D i m e t h y l m y l e r a n h a s s u b s e q u e n t l y b e e n s h o w n to r e a c t in v i v o b y a b i m o l e c u l a r p r o c e s s 5, t h o u g h t h e in v i v o m o d e of a c t i o n o f I M S r e m a i n s u n c e r t a i n .

A c o m p a r i s o n b e t w e n t h e m e t a b o l i s m of I M S a n d iso- p r o p y l i od ide ( I I I ) , w h i c h is k n o w n to r e a c t b y a bi- m o l e c u l a r p r o c e s s 6, r e v e a l s d i f f e r e n c e s in t h e i r in v i v o r e a c t i v i t i e s . I .p . a d m i n i s t r a t i o n o f 14C-IMS (75 m g / k g ) t o r a t s p r o d u c e d 2 u r i n a r y m e t a b o l i t e s , S-iso-propyi c y s t e i n e (V, R = H ) a n d t h e c o r r e s p o n d i n g m e r c a p t u r i c ac id , S-iso-propyl c y s t e i n e - N - a c e t a t e (V, R = C O C H a ) a m o u n t i n g t o 3 0 % of t h e a d m i n i s t e r e d dose . T h i s c o n t r a s t s w i t h t h e m e t a b o l i s m of ~C-iso-propyl i o d i d e (88 m g / k g , i .p . ) , in w h i c h o n l y t r a c e a m o u n t s ( 2 - 3 % of t h e dose) of t h e c y s t e i n e c o n j u g a t e (V, R = H ) w e r e d e t e c t e d in t h e u r i n e , a n d iso-propyl b r o m i d e , w h i c h is r e p o r t e d 7 to g i v e r i se to n o d e t e c t a b l e s u l p h u r - c o n t a i n i n g r e c t a - bo l i t e s .

1 C. D. R. DUNN and L. A. ELSON, Eur. J. d im biol. Res. 15, 771 (1970).

2 W. C. J. Ross, Biological Alkylating Agents (Butterworths, London 1962), p. 112.

3 H. JACKSON, B. W. FOX and A. W. CRAIO, J. Reprod. Fert. 2, 447 (1961).

a L. A. ELSON, Br. J. Haemat. 1,104 (1955). A. R. JONES, Chem. Biol. Interae. 6, 47 (1973).

6 R. L. HEPPOLETTE and R. E. ROBERTSON, Can. J. Chem. 44, 677 (1966).

7 ]~. A. BARNSLEY, T, H. GRENBY and L. YOUNG, Bioehem, J. 100, 282 (1966).

Excretion of radioactivity iu the urine and expired gases over 24 h from rats receiving equi-molar doses of compounds

Compound Dose Urine Carbon (mg/kg) dioxide

14C-iso-propy1 methanesulphonate 75 38 20

a~S-iso-propyl methanesulphonate 75 100 --

8~S-methanesulphonie acid 50 100 -- l~C-iso-propyl iodide 88 10 3

14C-iso-propyl alcohol 32 10 5

Activity, expressed as percent of the administered dose, was assessed as previously described 15. 14C-iso-propyl iodide and 14C-iso-propyI alcohol were obtained from the Radiochemical Centre, Amersham. 14C-IMS was prepared from l~C-iso-propyl alcohol and methane- sulphonyl chloride in pyridine at - - 5 ~ and asS-IMS from iso-propyI alcohol and silver aSS-methanesulphonate (Radiochemical Centre, Amersham) by the method of EM~ONS and FERRIS 16. aSS-methane- sulphonie acid was obtained by base hydrolysis of 35S-IMS. Com- pounds were administered i.p. as suspensions in arachis oil, except for iso-propyl alcohol and methanesulphordc acid, which were given in aqueous solution.

CH3SO2OH

ICH 3 ] CH 3 NHR CH cH-~176 CH+I >.-S-CH2- HOOOH CH 3 LCH 3 J CH3

CH3\ CH~ CH3\ /CH-1 ~" /CH-OH-~ /C=O--~ CO2

CH3 CH 3 CH 3

Fig. 2. Major metabolic pathways o~ I1V[S and iso-propy[ iodide in the rat. The cysteine conjugates were isolated from urine as previously described 18 and their chromatographic mobilities compared with authentic compoundsL On silica gel G plates (0.1 mm) in butanol giacial acetic acid water (4:2:1), the Rf values were 0.63 for (V) R=H and 0.86 for (V) R=COCH~. The metabolites were inter- converted either by acetylation (acetic anhydride) or hydrolysis (5N I:IC1 at 95~ for l h , or acylase at pH 7.4 and 37~ for 3 h), as well as being oxidised to S-iso-propyl-cysteine-S-oxide (Rf 0.42). Methanesulphonie acid (Rf 0.27) was identified by gas-liquid chromatography as the methyl ester 14.

15.5. 1973 Specialia 539

Apar t f rom detoxi f ica t ion by the a lkyla t ion of thiol groups, some degree of hydrolys is mus t occur, since nei ther I M S nor iso-propyl iodide are excre ted unchanged, and the excre t ion of methanesu lphonic acid ~rom 3~S-IMS paral lels t h a t of asS-methanesulphonic acid itself (Table). The hydrolys is product , iso-propyl aIcohol (VI), and its me tabo l i t e s acetone, were not detected f rom these com- pounds bu t as bo th iso-propyl alcohol and acetone are par t ia l ly oxidised in v ivo 9, i t is probable t h a t the expired ~dC-carbon dioxide (Table) represents this hydro ly t ic pa thway .

As the excre t ion pa t te rns of iso-propyl iodide and iso- propyl alcohol are a lmos t identical , hydrolysis of the former p robab ly represents the major de toxi f ica t ion route, a lkyla t ion react ions such as conjuga t ion wi th cysteine (glutathione) represent ing only a minor pa th- way. The different pa t t e rn of excre t ion of r ad ioac t iv i ty f rom IMS indicates , that a l though in v ivo hydrolysis is rapid (half-life at 37 ~ is 13 mill a t p i t 7) ~, the a lkyla t ion react ion is a major p a t h w a y (Figure 2). This can be inter- pre ted as react ion of the compounds by 2 different mechanisms; b imolecular for iso-propyl iodide (and pre- sumably the bromide) and unimolecular for IMS. The produc t ion of the h igh ly reac t ive d ime thy lca rbon ium ion (IV) f rom IMS by a unimolecular react ion is consistent wi th a rapid degree of a lkyla t ion both in ti le detoxif ica- t ion route and in the react ion of IMS wi th D N A in vi t ro to.

Whereas the different biological actions of a lkyla t ing agents have been a t t r ibu ted to thei r mechanisms of a lky la t ion ~, present and recent 1~ studies indicate that , at least for me thanesu lphona te esters, this m a y no t be true. IMS and d i m e t h y l m y l e r a n react by different mechanisms yet possess s imilar biological act ivi t ies sugges-

t ing tha t in some instances, the mechanism of a lkyla t ion m a y not be an i m p o r t a n t factor in thei r mode of action.

Zusammen/assung, Nachweis, dass die im R a t t e n h a r n auf t re tenden Metabol i ten Isopropyl jodid und Isopropyl- methansu l fona t fiir einen unterschiedl ichen Wirkungs- mechanismus der Substanzen in v ivo sprechen. Aus Iso- p ropy lmethansu l fona t en ts teh t du tch einen mono- molekularen Prozess das &usserst react ive Dimethy l - carboniumion, w~thrend Isopropyl jodid aufgrund einer b imolekularen Reak t ion haups~chl ich du tch Hydro lyse entgi f te t wird.

A. R. JONES and K. EDWARDS

Pharmacology Department, The University, Manchester M13 9 PT. , and Paterson Laboratories, Christie Hospital, Manchester M 20 9 BX . (England), 3 November 1972.

8 S. NAITO, Jap. J. Pharmae. 50, 578 (1954) ; Chem. Abstr, 52, 13918g I1958).

9 T. D. PRIC~ and D. RITTENEER% J. biol. Chem. 185, 449 (1950). 10 S. WALLES, Aeta chem. scand. 2d, 2012 (1970). 11 G. P. WARWICK, Cancer Res. 23, 1315 (1963). 12 A. R. JONES and I. S. C. CAMPBELL, Bioehem. Pharmae. 27, 2811

(1972). la K. EDWARDS and A. R. JONES, Biochem. Pharmac. 20, 1781 (1971). 14 •. EDWARDS, H. JACKSON and A. R. JONES, Biochem. Pharmac.

19, 1791 (1970). 15 K. EDWARDS, H. JACKSON and A. R. JONES, Biochern. Pharmac.

19, 1783 (1970). 16 W. D. E.~MONS and A. F. FERRIS, J. Am. chem. Sot. 75, 2257 (1953).

A c c e l e r a t i o n of Red Cell G l y c o l y s i s by Citrate due to In trace l lu lar pH E n h a n c e m e n t

Since the in t roduct ion of sodium ci t ra te for blood preserva t ion 1, i t has been used as all impor t an t ingredient for blood preserva t ion med ia : acid dextrose c i t ra te (ACD) and c i t ra te phospha te dextrose (CPD) solutions. Ci t ra te has been added to preservation media as an anticoagulant

and the effect of the anion on red cells has not been thoroughly studied. Although citrate anion is known to

oJ

o oContro I

6'.6 710 714 718 8'.2 pile

Fig. 1. The pH-curves of lactate formation in red cells in presence and absence of 33 mM citrate.

b e prac t ica l ly impermeable to red cell membrane 2, its high concent ra t ion is expected to exer t some influence on red cell metabol ism. Recen t ly we have found tha t the intracel lular p H (pH,) of red blood cells s tored in ACD medium is higher than the ext racel lu lar p H (pH,) of the suspension s. This f inding urged us to s tudy the glycolysis of red cells ill the presence of ci trate.

Methods. One-day-old ACD blood was obta ined f rom a local blood bank and red cells were washed thoroughly wi th isotonic saline. The cells were suspended ill a solut ion (120 m M NaC1, 5 m M KC1, 1 m M MgC12, 1 m M inorganic phospha te and 10 m M glucose) and incubated a t 37~ Dur ing incubat ion, the p H was kept cons tan t by a pH-s t a t wi th the addi t ion of 0.2 M NaOt t . Af te r 2 h preincubat ion, sodium c i t ra te solut ion was added to a final concent ra t ion of 33 m M and incuba ted for fur ther 2 h. Samples were t aken out a t in tervals for analyses.

Results and discussion. More t h a n 20% increase of the lac ta te format ion was observed when c i t ra te was added to the cell suspension a t p H 7.4. The increase by the c i t ra te addi t ion depended on the p H of the suspension as shown in Figure 1. Shif t of the p H curve was observed by the addi t ion of ci trate, which suggests the increase of the intracel lular pH. The possibi l i ty of p H increase inside the cells was fur ther suppor ted by the changes of the gly- colyt ic in termedia tes , t Iexose monophospha tes decreased

i p. Roos and J. R. TURNER, J. exp. Mcd. 23, 219 (1916). 2 L. GARBY, Folia haemat. 78, 295 (1961). 3 S. TSUDA, K. KAKINUMA and S. MINAKAI~II, Experientia 28, 1481

(1972).